The invention relates to animals in which the MutS homolog 5 (MSH5) gene is misexpressed and methods of using such animals or cells derived therefrom, e.g., in methods of evaluating fertility treatments.
MutS homolog 5 (MSH5) is a member of a family of proteins that are known to be involved in DNA mismatch repair (Modrich, P. and Lahue (1996) Annu. Rev. Biochem. 65, 101-133; Kolodner, R. (1996) Genes Dev. 10, 1433-1442). Germ line mutations in MSH2, MLH1 and MSH6 cause hereditary non-polyposis colon cancer (HNPCC) or Lynch syndrome (Leach, F. S. et al. (1993) Cell 75, 1215-1225; Bronner, C. E. et al. (1994) Nature 368, 258-261; Papadopoulos, N. et al. (1994) Science 263, 1625-1629; Akiyama, Y. et al. (1997) Cancer Res. 57, 3920-3923; Miyaki, M. et al. (1997) Nature Genet. 17, 271-272). Inactivation of Msh2, Mlh1, Msh6 and Pms2 in mice leads to hereditary predisposition to intestinal and other cancers (de Wind, N.et al. (1995) Cell 82, 321-330; Reitmair, A. H. et al. (1995) Nature Genet. 11, 64-70). Early studies in yeast revealed a role for some of these proteins, including MSH5, in meiosis (Hollingsworth, N. M., et al. (1995) Genes and Development 9, 1728-1739; Ross-Macdonald, P. and Roeder, G. S. (1994) Cell 79, 1069-1080). Gene targeting studies in mice confirmed roles for MLH1 and PMS2 in mammalian meiosis (Baker, S. M. et al. (1995) Cell 82, 309-320; Edelmann, W. et al. (1996) Cell 85, 1125-1134; Baker, S. M. et al. Nature Genet. 13, 336-342).
The present invention is based, at least in part, on the generation of animals which are homozygous for a null mutation in the MutS homolog 5 (MSH5) gene and the observation that these animals are sterile. Accordingly, the invention features, a non-human animal, in which the gene encoding the MutS homolog 5 (MSH5) protein is misexpressed.
In preferred embodiments the animal, which is preferably a transgenic animal, is a mammal, e.g., a non human primate or a swine, e.g., a miniature swine, a monkey, a goat, or a rodent, e.g., a rat, but preferably a mouse.
In preferred embodiments, expression of the gene encoding the MSH5 protein is decreased as compared to the wild-type animal. For example, the levels of the MSH5 protein can be suppressed by, at least, 50%, 60%, 70%, 80%, 90%, or 100% as compared to the wild-type animal.
In preferred embodiments, misexpression of the gene encoding the MSH5 protein is caused by disruption of the MSH5 gene. For example, the MSH5 gene can be disrupted through removal of DNA encoding all or part of the protein.
In preferred embodiments, the animal can be heterozygous or homozygous for a misexpressed MSH5 gene, e.g., it can be a transgenic animal heterozygous or homozygous for an MSH5 transgene.
In preferred embodiments, the animal is a transgenic mouse with a transgenic disruption of the MSH5 gene, preferably an insertion or deletion, which inactivates the gene product.
In another aspect, the invention features, a nucleic acid molecule which, when introduced into an animal or cell, results in the misexpression of the MSH5 gene in the animal or cell. In preferred embodiments, the nucleic acid molecule, includes an MSH5 nucleotide sequence which includes a disruption, e.g., an insertion or deletion and preferably the insertion of a marker sequence. For example, a nucleic acid molecule can be the targeting construct shown in FIG. 1.
In another aspect, the invention features, a method of evaluating a fertility treatment. The method includes: administering the treatment to an MSH5 misexpressing animal, e.g., a transgenic mouse, or a cell therefrom; and determining the effect of the treatment on a fertility indication, e.g., sperm count, testicular size, or oocyte morphology, to thereby evaluate the treatment for fertility. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein. In other preferred embodiments, the method uses a transgenic mouse in which the expression of the MSH5 gene is inhibited. In yet other preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the MSH5 gene is inhibited.
In another aspect, the invention features, a method for identifying a compound which modulates the activity of MSH5. The method includes contacting MSH5 with a test compound and determining the effect of the test compound on the activity of MSH5 to, thereby, identify a compound which modulates MSH5 activity. In preferred embodiments, the activity of MSH5 is inhibited.
In another aspect, the invention features, a method for modulating the activity of MSH5. The method includes contacting MSH5 or a cell expressing MSH5 with a compound which binds to MSH5 in an amount sufficient (e.g., a sufficient concentration) to modulate the activity of MSH5. In preferred embodiments, the activity of MSH5 is inhibited, e.g., the method can be used in contraception.
In another aspect, the invention features, a method of identifying a subject having or at risk of developing a fertility disease or disorder. The method includes obtaining a sample from said subject; contacting the sample with a nucleic acid probe or primer which selectively hybridizes to MSH5 and determining whether aberrant MSH5 expression or activity exists in the sample, thereby, identifying a subject having or at risk of developing a fertility disease or disorder.
In another aspect, the invention features, an isolated cell, or a purified preparation of cells, from an MSH5 misexpressing animal, e.g., an MSH5 misexpressing animal described herein. In preferred embodiments, the cell is a transgenic cell, in which the gene encoding the MSH5 protein is misexpressed. The cell, preferably a transgenic cell can be an oocyte or a spermatocyte.
In preferred embodiments, the cell is heterozygous or homozygous for the transgenic mutant gene.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.